The long-term objectives pf this renewal application continue to focus on developing a Comprehensive understanding of the physiology and pathophysiology of the intestinal absorption process of the water-soluble vitamin B1 (thiamine) at the cellular and molecular levels, how the process is regulated, and how it is affected by external factors like chronic alcohol exposure. Thiamine is indispensable for normal human health arid is obtained from exogenous sources via intestinal absorption. Studies during the current funding period have used Slc19a2 -/-and Slc 9a3 -/- knockout mouse models to show that both thiamin transporter 1 & 2 (THTR-1 & 2) are involved in intestinal thiamin absorption; that the intestinal thiamine uptake process is adaptively regulated by extracellular substrate level via transcriptional mechanism involving the transcriptional factor SP1; that tetraspanin-1 (Tspan-1) and transmembrane 4 super-family member 4 (TM4SF4) proteins are interacting partners with intestinal THTR-1 and THTR-2, respectively and that they affect their physiology/cell biology; and that enteropathogenic Escherichia coli and enterotoxigenic E. Coli inhibit intestinal thiamine uptake. Two additional and very relevant studies were also initiated during the current funding period with the first dealing with the identification of existence of a specific and efficient carrier-mediated system for uptake of the microbiota-generated thiamin pyrophosphate (TPP) in the colon (i.e., the SLC44A4 system), and the second is the demonstration that the inhibitory effect of chronic alcohol feeding/exposure on intestinal thiamine uptake is mediated at the level of transcription of theSLC19A2 and SLG19A3: genes. Based on these new findings, our working hypotheses during the next period will be that the SLC44A4 system is a specific and regulated colonic TPP uptake system, and that transcriptional (e. g., epigenetic) mechanisms are involved in mediating the inhibitory effect of chronic alcohol exposure on intestinal thiamin uptake. Four specific aims are proposed to address these hypotheses, and will utilize state-of-the-art cellular/molecular approaches. Results of these studies should continue to provide novel information regarding the physiology/pathophysiology of the intestinal vitamin B1 absorption process.